International Journal of Molecular Sciences Review N2A Titin: Signaling Hub and Mechanical Switch in Skeletal Muscle Kiisa Nishikawa 1,* , Stan L. Lindstedt 1, Anthony Hessel 2 and Dhruv Mishra 1 1 Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640, USA; [email protected] (S.L.L.); [email protected] (D.M.) 2 Institute of Physiology II, University of Münster, 48149 Münster, Germany; [email protected] * Correspondence: [email protected]; Tel.: +1-928-523-9497 Received: 12 May 2020; Accepted: 1 June 2020; Published: 1 June 2020 Abstract: Since its belated discovery, our understanding of the giant protein titin has grown exponentially from its humble beginning as a sarcomeric scaffold to recent recognition of its critical mechanical and signaling functions in active muscle. One uniquely useful model to unravel titin’s functions, muscular dystrophy with myositis (mdm), arose spontaneously in mice as a transposon-like LINE repeat insertion that results in a small deletion in the N2A region of titin. This small deletion profoundly affects hypertrophic signaling and muscle mechanics, thereby providing insights into the function of this specific region and the consequences of its dysfunction. The impact of this mutation is profound, affecting diverse aspects of the phenotype including muscle mechanics, developmental hypertrophy, and thermoregulation. In this review, we explore accumulating evidence that points to the N2A region of titin as a dynamic “switch” that is critical for both mechanical and signaling functions in skeletal muscle. Calcium-dependent binding of N2A titin to actin filaments triggers a cascade of changes in titin that affect mechanical properties such as elastic energy storage and return, as well as hypertrophic signaling. The mdm phenotype also points to the existence of as yet unidentified signaling pathways for muscle hypertrophy and thermoregulation, likely involving titin’s PEVK region as well as the N2A signalosome. Keywords: mechano-sensing; muscle hypertrophy; muscle mechanics; muscular dystrophy; striated muscle; titinopathy 1. Introduction: A Small Change in A Giant Protein Leads to Severe Titinopathy in mdm Titinopathies [1] are inherited diseases of skeletal and cardiac muscle that are caused by mutations in the Ttn gene, which encodes for titin, the largest known protein [2]. Titin is the third most abundant protein in the muscles of vertebrates [3], and spans an entire half-sarcomere (1 µm) from the M-line to the Z-disk [4]. Titin plays many important roles in striated muscle, including passive force generation [5], maintenance of sarcomere integrity [6], and myofibrillar assembly [4,7]. Due to its large size and repetitive sequence, the Ttn gene exhibits enormous variability among humans [8,9]. Millions of potential isoforms are possible due to alternative splicing of the many (>360) Ttn exons [10]. Most of the disease-associated Ttn variants include mutations with large effects on the expressed titin protein, including nonsense, missense, and truncating mutations, insertions/deletions, and splice mutations [11]. In affected individuals, compound heterozygosity is common [12]. Despite the relatively large effects of these mutations on the expressed titin protein, many titin mutations are associated with relatively late onset myopathy and/or cardiomyopathy [11]. The diverse mechanisms of post-transcriptional and post-translational modification, and the diversity of signaling functions already described for this giant protein are staggering in number and complexity [13], which may help to explain why Int. J. Mol. Sci. 2020, 21, 3974; doi:10.3390/ijms21113974 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3974 2 of 18 the underlying mechanisms through which titin mutations produce muscle disease remain largely unknown [14]. In contrast to more typical titinopathies, muscular dystrophy with myositis (mdm) in mice [15,16], among the earliest identified titinopathies [1,17], paradoxically presents a severe phenotype that is caused by a small deletion. Just 83 amino acids are missing from the giant titin protein [15], the largest isoform of which contains 38,000 amino acids. This represents a miniscule fraction (0.2%) of the entire protein. The mdm deletion is located at the N2A-PEVK border of I-band titin (Figure1A). The N2A region of titin (Figure1B) is comprised of four Ig domains and a unique insertion sequence (UN2A) in the order Ig80-UN2A-Ig81-Ig82-Ig83 [18]. In mdm, 21 amino acids are deleted from Ig83, and the remaining 61 amino acids are deleted from linking and PEVK regions (Figure1B). Given the small size of the deletion, mdm is a surprisingly severe titinopathy with early onset shortly after birth and progressive degeneration, leading to early death [19]. Although the primary deletion is small, it remains to be determined whether splicing of the Ttn gene itself might also be affected. The severity of the phenotype suggests that this small region of titin plays a critical role in muscle D112 158 function. A transgenic TTN − mouse was recently developed [20] in which 47 exons from the N-terminus of the PEVK region were deleted, including 28 amino acids at the C-terminus of the mdm deletion, yet life span and muscle function of these mice are normal [20]. Thus, it appears likely that only the remaining 55 amino acids deleted from Ig83 and the N2A-PEVK linker region are responsible for the severe mdm phenotype. Figure 1. (A) Layout of titin in skeletal muscle sarcomeres. Each titin molecule is bound to the thin filament (blue) in the Z-disk (blue triangles on left side of half-sarcomere) and to the thick filaments (purple) in the A-band. The N2A segment (red) is located between the proximal tandem Ig segments (orange) and the PEVK segment (green). Ig, immunoglobulin. Reproduced with permission from Nishikawa [21]. Copyright 2016, The Company of Biologists Ltd. (B) Layout of the N2A region. This region includes Ig80, unique N2A sequence (UN2A), Ig81, Ig82, and Ig83. The exons of the mouse Ttn gene are shown below, along with the location of the mdm deletion. Lane [16] first reported the mdm mutation, which arose spontaneously on the C57BJ/6j mouse background at the Jackson Laboratories. Although the mdm mutation was initially mapped to chromosome 2, the affected gene(s) remained unknown [16]. When chromosome 2 was identified as the location of titin and nebulin genes [22], the hunt for the mdm mutation was quickly focused on Int. J. Mol. Sci. 2020, 21, 3974 3 of 18 these genes. Müller-Seitz et al. [22] collected titin and nebulin cDNA from mdm muscle and probed different regions for genetic mutations; however, no changes in titin or nebulin cDNA were uncovered. Nearly a decade later, with quickly advancing technology in sequencing, the site of the mutation was finally located within the titin gene [15]. Mdm is recessive lethal and first manifests in development as a kyphosis of the spine in homozygous mice at 12 days after birth [16]. Mdm mice exhibit a complex phenotype that, in addition to severe kyphosis, includes reduced body mass [19], rigid gait, and early death at approximately 60 days of age [15]. Histologically, signs of muscle degeneration appear in the soleus muscle within the first two weeks of birth, and 1–2 weeks later in the tibialis anterior [19]. However, regeneration of fibers is also evident from the presence of central nuclei in mdm muscles and the increased frequency and activity of satellite cells starting at 40 days of age, compared to wild type muscles [19]. The mdm mutation affects muscle function from myofibril to intact muscle [23]. Fiber bundles from mdm soleus have normal passive and active force at 24 days of age [24]. Single myofibrils have normal passive force, and only a small deficit in active isometric force at optimal length [25]. Fiber bundles have normal length- and calcium dependence of force [26], but also have increased passive tension and psoas fibers have reduced isometric tension compared to wild type muscles [27], although isometric tension is spared in extensor digitorum longus (EDL) fibers [26]. Intact muscles from mdm mice generally exhibit lower isometric tension, but the effect is greater in soleus than EDL [28]. In addition to changes in muscle structure and function, several recent studies demonstrate that the mdm mutation also affects post-natal growth and thermoregulation. While Witt et al. [24] assumed that mdm mice lose weight after birth, other studies suggest that they actually fail to grow after weaning. Huebsch et al. [29] found that skeletal muscle fibers from mdm mice remained small in diameter at post-natal day 14 when normal fibers have already undergone significant developmental hypertrophy. Pace et al. [30] also found that body mass failed to increase in mdm mice after weaning. Compared to wild type littermates, the growth of the long bones of the hindlimbs was also reduced after birth, although limb proportions remained relatively normal, indicating that growth of the skeleton is also reduced in mdm mice. These observations suggest impairment of normal hypertrophic signaling in skeletal muscles after birth, as well as concomitant changes in bone growth commonly observed in neuromuscular disease [31]. Two recent studies also demonstrate impaired thermoregulation in mdm mice. Taylor-Burt et al. [32] found that mdm mice failed to maintain a constant body temperature at ambient temperatures ranging from 20 to 37 ◦C, in contrast to wild type littermates. Homozygous mdm mice exhibited shivering thermogenesis